THERAPY GUIDANCE AND/OR THERAPY MONITORING FOR TREATMENT OF SHOCK

Abstract

Subject matter of the present invention is a method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock, wherein said method is comprising the steps: determining the level of DPP3 in a sample of bodily fluid of said subject; comparing said level of determined DPP3 to a predetermined threshold,
wherein said subject is predicted to run into refractory shock or is diagnosed as having refractory shock if said determined level of DPP3 is above said predetermined threshold.

Further subject matter relates to vasopressors, angiotensin-receptor agonists and/or precursors thereof, inhibitors of the activity of DPP3 and anti-ADM antibodies for use in therapy of shock in a subject that either runs into shock or that has developed shock.

Claims

1. A method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock, wherein said method comprising determining the level of DPP3 in a sample of bodily fluid of said subject; and comparing said level of determined DPP3 to a predetermined threshold, wherein said subject is predicted to run into refractory shock or is diagnosed as having refractory shock if said determined level of DPP3 is above said predetermined threshold.

2. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein said shock is selected from the group comprising shock due to hypovolemia, cardiogenic shock, obstructive shock and distributive shock, in particular cardiogenic shock or septic shock.

3. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein said shock is a vasopressor-resistant shock.

4. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein in case of cardiogenic shock said subject may have suffered an acute coronary syndrome (e.g. acute myocardial infarction) or wherein said subject has heart failure (e.g. acute decompensated heart failure), myocarditis, arrhythmia, cardiomyopathy, valvular heart disease, aortic dissection with acute aortic stenosis, traumatic chordal rupture or massive pulmonary embolism, or in case of hypovolemic shock said subject may have suffered a hemorrhagic disease including gastrointestinal bleed, trauma, vascular etiologies (e.g. ruptured abdominal aortic aneurysm, tumor eroding into a major blood vessel) and spontaneous bleeding in the setting of anticoagulant use or a non-hemorrhagic disease including vomiting, diarrhea, renal loss, skin losses/insensible losses (e.g. burns, heat stroke) or third-space loss in the setting of pancreatitis, cirrhosis, intestinal obstruction, trauma, or in case of obstructive shock said patient may have suffered a cardiac tamponade, tension pneumothorax, pulmonary embolism or aortic stenosis, or in case of distributive shock said patient may have septic shock, neurogenic shock, anaphylactic shock or shock due to adrenal crisis.

5. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein said method is used for initiation and/or termination and/or stratification and/or guidance of treatment.

6. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein a treatment is initiated and/or maintained and/or withheld and/or terminated if said determined level of DPP3 is above said predetermined threshold.

7. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 6, wherein said treatment is selected from the group of vasopressors, Angiotensin-Receptor-Agonists and/or precursors thereof, inhibitors of the DPP3 activity and anti-adrenomedullin antibodies or anti-adrenomedullin antibody fragments.

8. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein either the level of DPP3 protein and/or the level of active DPP3 is determined and compared to a predetermined threshold, wherein the level of DPP3 is determined by contacting said sample of bodily fluid with a capture binder that binds specifically to DPP3.

9. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein a treatment with Angiotensin-Receptor-Agonists and/or precursors thereof and/or inhibitors of the DPP3 activity is initiated and/or continued when the level of DPP3 in said sample is above a certain threshold and/or wherein a treatment with vasopressors is withheld and/or terminated if said determined level of DPP3 is above said predetermined threshold.

10. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 1, wherein a treatment with vasopressors is initiated and/or continued when the level of DPP3 in said sample is below a certain threshold and/or wherein a treatment with Angiotensin-Receptor-Agonists and/or precursors thereof and/or inhibitors of the DPP3 activity is withheld and/or terminated if the said determined level of DPP3 is below said predetermined threshold.

11. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 9, wherein in addition the level of Pro-adrenomedullin or fragments thereof is determined and wherein treatment with an anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of Pro-adrenomedullin or fragments thereof in said sample is above a certain threshold and/or wherein a treatment with an anti-ADM antibody or anti-ADM antibody fragment is withheld and/or terminated if the said determined level of Pro-adrenomedullin or fragments thereof is below said predetermined threshold.

12. The method for predicting or diagnosing a refractory shock in a subject that either runs into shock or that has developed shock according to claim 9, wherein treatment with an anti-ADM antibody or anti-ADM antibody fragment and/or Angiotensin-Receptor-Agonists and/or precursors thereof and/or inhibitors of the DPP3 activity is initiated and/or continued if the level of Pro-adrenomedullin or fragments thereof in said sample is above a certain threshold and said determined level of DPP3 is above said predetermined threshold of DPP3.

13. A vasopressor for use in therapy of shock in a subject that either runs into shock or that has developed shock, wherein said subject has a level of DPP3 in a sample of bodily fluid of said subject that is below a predetermined threshold, when determined by a method according to claim 1.

14. An inhibitor of the activity of DPP3 for use in therapy of shock in a subject that either runs into shock or that has developed shock, wherein said subject has a level of DPP3 in a sample of bodily fluid of said subject that is above a predetermined threshold when determined by a method according to claim 1, wherein the inhibitor of the activity of DPP3 is selected from the group comprising anti-DPP3 antibody or anti-DPP3 antibody fragment or anti-DPP3 non-Ig scaffold.

15. The inhibitor of the activity of DPP3 for use in therapy of shock in a subject that either runs into shock or that has developed shock according to claim 14, wherein said inhibitor is administered in combination with an Angiotensin-Receptor-Agonist and/or precursor thereof.

16. The inhibitor of the activity of DPP3 for use in therapy of shock in a subject that either runs into shock or that has developed shock according to claim 15, wherein said Angiotensin-Receptor-Agonist and/or precursor thereof is selected from the group comprising angiotensin I, angiotensin II, angiotensin III, angiotensin IV, in particular angiotensin II.

17. A method of treatment of shock in a subject that either runs into shock or that has developed shock, the method comprising administering vasopressor to said subject, wherein said subject has a level of DPP3 in a sample of bodily fluid of said subject that is below a predetermined threshold, when determined by a method according claim 1.

18. A method of treatment of shock in a subject that either runs into shock or that has developed shock, the method comprising administering inhibitor of DPP3 activity to said subject, wherein said subject has a level of DPP3 in a sample of bodily fluid of said subject that is above a predetermined threshold when determined by a method according to claim 1.

19. The method of treatment of shock in a subject that either runs into shock or that has developed shock according to claim 18, the method comprising administering an inhibitor of DPP3 activity to said subject, wherein said inhibitor is administered in combination with an Angiotensin-Receptor-Agonist and/or a precursor thereof.

20. The method of treatment of shock in a subject that either runs into shock or that has developed shock according to claim 17, the method comprising administering Angiotensin-Receptor-Agonists and/or precursors thereof and/or inhibitor of DPP3 activity to said subject, wherein the treatment with said Angiotensin-Receptor-Agonists and/or inhibitors of the DPP3 activity is initiated and/or continued when the level of DPP3 in a sample of said subject is above a certain threshold and/or wherein a treatment with vasopressors is withheld and/or terminated if said determined level of DPP3 is above said predetermined threshold.

21. The method of treatment of shock in a subject that either runs into shock or that has developed shock according to claim 17, the method comprising administering vasopressor to said subject, wherein the treatment with said vasopressors is initiated and/or continued when the level of DPP3 in a sample of bodily fluid of said subject is below a certain threshold and/or wherein a treatment with Angiotensin-Receptor-Agonists and/or precursors thereof and/or inhibitors of the DPP3 activity is withheld and/or terminated if the said determined level of DPP3 is below said predetermined threshold.

22. The method of treatment of shock in a subject that either runs into shock or that has developed shock according to claim 19, the method comprising administering Angiotensin-Receptor-Agonists and/or precursors thereof and/or inhibitor of DPP3 activity to said subject, and wherein in addition the level of Pro-adrenomedullin or fragments thereof is determined and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of Pro-adrenomedullin or fragments thereof in said sample is above a certain threshold and/or wherein a treatment with an anti-ADM antibody or anti-ADM antibody fragment is withheld and/or terminated if the said determined level of Pro-adrenomedullin or fragments thereof is below said predetermined threshold.

23. The method of treatment of shock in a subject that either runs into shock or that has developed shock according to claim 20, the method comprising administering vasopressor to said subject, and wherein in addition the level of Pro-adrenomedullin or fragments thereof is determined and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of Pro-adrenomedullin or fragments thereof in said sample is above a certain threshold and/or wherein a treatment with an anti-ADM antibody or anti-ADM antibody fragment is withheld and/or terminated if the said determined level of Pro-adrenomedullin or fragments thereof is below said predetermined threshold.

24. The method of treatment of shock in a subject that either runs into shock or that has developed shock according to claim 21, the method comprising administering anti-ADM antibody or anti-ADM antibody fragment to said subject, wherein in addition the level of Pro-adrenomedullin or fragments thereof is determined and wherein treatment with said anti-ADM antibody or anti-ADM antibody fragment is initiated and/or continued when the level of Pro-adrenomedullin or fragments thereof in said sample is above a certain threshold and/or wherein a treatment with an anti-ADM antibody or anti-ADM antibody fragment is withheld and/or terminated if the said determined level of Pro-adrenomedullin or fragments thereof is below said predetermined threshold.

25. A method for prognosing an outcome and/or the risk of an adverse event in a subject that has developed refractory shock, wherein said method comprising: determining the level of DPP3 in a sample of bodily fluid of said subject; comparing said level of determined DPP3 to a predetermined threshold; and correlating said level of DPP3 with said risk of an adverse event in said subject, wherein an elevated level above a certain threshold is predictive for an enhanced risk of said adverse events or, correlating said level of DPP3 with success of a therapy or intervention in said subject, wherein a level below a certain threshold is predictive for a success of therapy or intervention.

Description

EXAMPLES

Example 1—Methods for the Measurement of DPP 3 Protein and DPP3 Activity

[0368] Generation of antibodies and determination DPP3 binding ability: Several murine antibodies were produced and screened by their ability of binding human DPP3 in a specific binding assay (see Table 1).

Peptides/Conjugates for Immunization:

[0369] DPP3 peptides for immunization were synthesized, see Table 1, (JPT Technologies, Berlin, Germany) with an additional N-terminal cystein (if no cystein is present within the selected DPP3-sequence) residue for conjugation of the peptides to Bovine Serum Albumin (BSA). The peptides were covalently linked to BSA by using Sulfolink-coupling gel (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio. Recombinant GST-hDPP3 was produced by USBio (United States Biological, Salem, Mass., USA).

Immunization of mice, immune cell fusion and screening:

[0370] Balb/c mice were intraperitoneally (i.p.) injected with 84 pg GST-hDPP3 or 100 pg DPP3-peptide-BSA-conjugates at day 0 (emulsified in TiterMax Gold Adjuvant), 84 μg or 100 μg at day 14 (emulsified in complete Freund's adjuvant) and 42 μg or 50 μg at day 21 and 28 (in incomplete Freund's adjuvant). At day 49 the animal received an intravenous (i.v.) injection of 42 μg GST-hDPP3 or 50 μg DPP3-peptide-BSA-conjugates dissolved in saline. Three days later the mice were sacrificed and the immune cell fusion was performed.

[0371] Splenocytes from the immunized mice and cells of the myeloma cell line SP2/0 were fused with 1 ml 50% polyethylene glycol for 30 s at 37° C. After washing, the cells were seeded in 96-well cell culture plates. Hybrid clones were selected by growing in HAT medium [RPMI 1640 culture medium supplemented with 20% fetal calf serum and HAT-Supplement]. After one week, the HAT medium was replaced with HT Medium for three passages followed by returning to the normal cell culture medium.

[0372] The cell culture supernatants were primarily screened for recombinant DPP3 binding IgG antibodies two weeks after fusion. Therefore, recombinant GST-tagged hDPP3 (USBiologicals, Salem, USA) was immobilized in 96-well plates (100 ng/well) and incubated with 50 μl cell culture supernatant per well for 2 hours at room temperature. After washing of the plate, 50 μl/well POD-rabbit anti mouse IgG was added and incubated for 1 h at RT. After a next washing step, 50 μl of a chromogen solution (3.7 mM o-phenylendiamine in citrate/hydrogen phosphate buffer, 0.012% H.sub.2O.sub.2) were added to each well, incubated for 15 minutes at RT and the chromogenic reaction stopped by the addition of 50 μl 4N sulfuric acid. Absorption was detected at 490 mm.

[0373] The positive tested microcultures were transferred into 24-well plates for propagation. After retesting the selected cultures were cloned and re-cloned using the limiting-dilution technique and the isotypes were determined.

Mouse Monoclonal Antibody Production

[0374] Antibodies raised against GST-tagged human DPP3 or DPP3-peptides were produced via standard antibody production methods (Marx et al. 1997) and purified via Protein A. The antibody purities were >90% based on SDS gel electrophoresis analysis.

Characterization of Antibodies—Binding to hDPP3 and/or Immunization Peptide

[0375] To analyze the capability of DPP3/immunization peptide binding by the different antibodies and antibody clones a binding assay was performed: [0376] a) Solid phase

[0377] Recombinant GST-tagged hDPP3 (SEQ ID NO. 1) or a DPP3 peptide (immunization peptide, SEQ ID NO. 2) was immobilized onto a high binding microtiter plate surface (96-Well polystyrene microplates, Greiner Bio-One international AG, Austria, 1 μg/well in coupling buffer [50 mM Tris, 100 mM NaCl, pH7.8], 1 h at RT). After blocking with 5% bovine serum albumin, the microplates were vacuum dried. [0378] b) Labelling procedure (Tracer)

[0379] 100 μg (100 μl) of the different antiDPP3 antibodies (detection antibody, 1 mg/ml in PBS, pH 7.4) were mixed with 10 μl acridinium NHS-ester (1 mg/ml in acetonitrile, InVent GmbH, Germany; EP 0 353 971) and incubated for 30 min at room temperature. Labelled antiDPP3 antibody was purified by gel-filtration HPLC on Shodex Protein 5 μm KW-803 (Showa Denko, Japan). The purified labeled antibody was diluted in assay buffer (50 mmol/1 potassium phosphate, 100 mmol/1 NaCl, 10 mmol/1 Na.sub.2-EDTA, 5 g/l bovine serum albumin, 1 g/l murine IgG, 1 g/l bovine IgG, 50 pmol/I amastatin, 100 pmol/I leupeptin, pH 7.4). The final concentration was approx. 5-7*10.sup.6 relative light units (RLU) of labelled compound (approx. 20 ng labeled antibody) per 200 μl. acridinium ester chemiluminescence was measured by using a Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). [0380] c) hDPP3 binding assay

[0381] The plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 2-4 h at 2-8° C. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG).

Characterization of Antibodies—hDPP3-Inhibition Analysis

[0382] To analyze the capability of DPP3 inhibition by the different antibodies and antibody clones a DPP3 activity assay with known procedure (Jones et al., 1982) was performed. Recombinant GST-tagged hDPP3 was diluted in assay buffer (25 ng/ml GST-DPP3 in 50 mM Tris-HCl, pH7.5 and 100 μM ZnCl.sub.2) and 200 μl of this solution incubated with 10 μg of the respective antibody at room temperature. After 1 hour of pre-incubation, fluorogenic substrate Arg-Arg-βNA (20 μl, 2 mM) was added to the solution and the generation of free βDNA over time was monitored using the Twinkle LB 970 microplate fluorometer (Berthold Technologies GmbH & Co. KG) at 37° C. Fluorescence of βNA is detected by exciting at 340 nm and measuring emission at 410 nm. Slopes (in RFU/min) of increasing fluorescence of the different samples are calculated. The slope of GST-hDPP3 with buffer control is appointed as 100% activity. The inhibitory ability of a possible capture-binder is defined as the decrease of GST-hDPP3 activity by incubation with said capture-binder in percent.

[0383] The following table represents a selection of obtained antibodies and their binding rate in Relative Light Units (RLU) as well as their relative inhibitory ability (%; table 1). The monoclonal antibodies raised against the below depicted DPP3 regions, were selected by their ability to bind recombinant DPP3 and/or immunization peptide, as well as by their inhibitory potential.

[0384] All antibodies raised against the GST-tagged, full length form of recombinant hDPP3 show a strong binding to immobilized GST-tagged hDPP3. Also antibodies raised against the SEQ ID NO.: 2 peptide bind to GST-hDPP3. The SEQ ID NO.: 2 antibodies also strongly bind to the immunization peptide.

TABLE-US-00005 TABLE 1 list of antibodies raised against full-length or sequences of hDPP3 and their ability to bind hDPP3 (SEQ ID NO.: 1) or immunization peptide (SEQ ID NO.: 2) in RLU, as well as the maximum inhibition of recombinant GST-hDPP3. immunization hDPP3 peptide Max. Sequence hDPP3 binding binding inhibition number Antigen/Immunogen region Clone [RLU] [RLU] of hDPP3 SEQ ID GST tagged recombinant FL-   1-737 2552 3.053.621 0 65% NO.: 1 hDPP3   2553 3.777.985 0 35% 2554 1.733.815 0 30% 2555 3.805.363 0 25% SEQ ID CETVINPETGEQIQSWYRSGE 474-493 1963 141.822 2.163.038 60% NO.: 2 1964 100.802 2.041.928 60% 1965 99.493 1.986.794 70% 1966 118.097 1.990.702 65% 1967 113.736 1.909.954 70% 1968 105.696 2.017.731 65% 1969 82.558 2.224.025 70%

[0385] The development of a luminescence immunoassay for the quantification of DPP3 protein concentrations (DPP3-LIA) as well as an enzyme capture activity assay for the quantification of DPP3 activity (DPP3-ECA) have been described recently (Rehfeld et al. 2018. JALM. in press), which is incorporated here in its entirety by reference.

Example 2—DPP3 for Prognosis of Short-Term Mortality

[0386] DPP3 concentration in plasma of patients with sepsis/septic shock and cardiogenic shock was determined and related to the short term-mortality of the patients.

Study Cohort—Sepsis/Septic Shock

[0387] In 574 plasma samples from patients of the Adrenomedullin and Outcome in Severe Sepsis and Septic Shock (AdrenOSS) study DPP3 was measured. AdrenOSS is a prospective, observational, multinational study including 583 patients admitted to the intensive care unit with sepsis or septic shock (Hollinger et al., 2018). 292 patients were diagnosed with septic shock.

Study Cohort—Cardiogenic Shock

[0388] Plasma samples from 108 patients that were diagnosed with cardiogenic shock were screened for DPP3. Blood was drawn within 6 h from detection of cardiogenic shock. Mortality was followed for 7 days.

Hdpp3 Immunoassay:

[0389] An immunoassay (LIA) or an activity assays (ECA) detecting the amount of human DPP3 (LIA) or the activity of human DPP3 (ECA), respectively, was used for determining the DPP3 level in patient plasma. Antibody immobilization, labelling and incubation were performed as described in Rehfeld et al. (Rehfeld et al. 2018).

Results

[0390] Short-term patients' survival in sepsis patients was related to the DPP3 plasma concentration at admission. Patients with DPP3 plasma concentration above 40.5 ng/mL (3rd Quartile) had an increased mortality risk compared to patients with DPP3 plasma concentrations below this threshold (FIG. 1A). Applying this cut-off to the subcohort of septic shock patients, revealed an even more pronounced risk for short-term mortality in relation to high DPP3 plasma concentrations (FIG. 1B). When the same cut-off is applied to patients with cardiogenic shock, also an increased risk for short-term mortality within 7 days is observed in patients with high DPP3 (FIG. 1C).

Example 3—Purification of Human Native DPP3

[0391] Human erythrocyte lysate was applied on a total of 100 ml of Sepahrose 4B resin (Sigma-Aldrich) and the flow through was collected. The resin was washed with a total of 370 mL PBS buffer, pH 7.4 and the wash fraction was combined with the collected flow through, resulting in a total volume of 2370 mL.

[0392] For the immuno-affinity purification step, 110 mg of monoclonal anti-hDPP3 mAb AK2552 were coupled to 25.5 mL of UltraLink Hydrazide Resin (Thermo Fisher Scientific) according to the manufacturer's protocol (GlycoLink Immobilization Kit, Thermo Fisher Scientific). The coupling efficiency was 98%, determined by quantification of uncoupled antibody via Bradford-technique. The resin-antibody conjugate was equilibrated with 10 bed volumes of wash-binding buffer (PBS, 0.1% TritonX-100, pH 7.4), combined with 2370 mL of cleared red blood cell lysate and incubated at 4° C. under continuous stirring for 2 h. Consequently, 100 mL of the incubation mixture was spread on ten 15 mL polypropylene columns and the flow-through was collected by centrifugation at 1000×g for 30 seconds. This step was repeated several times resulting in 2.5 mL of DPP3-loaded resin per column. Each column was washed times with 10 mL of wash-binding buffer using the gravity-glow approach. DPP3 was eluted by placing each column in 15-mL falcon tube containing 2 mL of neutralization buffer (IM Tris-HCl, pH 8.0), followed by addition of 10 mL of elution buffer (100 mM Glycine-HCl, 0.1% TritonX-100, pH 3.5) per column and immediate centrifugation for 30 seconds at 1000×g. The elution step was repeated 3 times in total resulting in 360 mL of combined eluates. The pH of the neutralized eluates was 8.0.

[0393] The combined eluates were loaded on a 5 mL HiTrap Q-sephare HP column (GE Healthcare) equilibrated with IEX-buffer A1 (100 mM Glycine, 150 mM Tris, pH 8.0) using the sample pump of the Äkta Start system (GE Healthcare). After sample loading, the column was washed with five column volumes of IEX Buffer A2 (12 mM NaH.sub.2PO.sub.4, pH 7.4) to remove unbound protein. Elution of DPP3 was achieved by applying a sodium chloride gradient over 10 column volumes (50 mL) in a range of 0-1 M NaCl using IEX-buffer B (12 mM NaH.sub.2PO.sub.4, 1 M NaCl, pH 7.4). The eluates were collected in 2 mL fractions. Buffers used for ion exchange chromatography were sterile filtered using a 0.22 μM bottle-top filter.

[0394] A purification table with the respective yields and activities of each purification step is given in table 2. FIG. 2 shows an SDS-PAGE on a gradient gel (4-20%) of native hDPP3 purified from human erythrocyte lysate.

TABLE-US-00006 TABLE 2 Purification of DPP 3 from human erythrocytes Total DPP3 Total activity in Specific amount in protein in μmol/min Yield.sup.d) in activity in Purification Step % (LIA).sup.a) mg.sup.b) (ECA).sup.c) % U/mg.sup.e) factor.sup.f) Lysate 100 204160 55 100 0.00027 — IAP 80.6 71.2 46.1 84 0.65 2407 IEX 75 6.6 38.7 70 5.9 21852 .sup.a)Relative DPP3 amount was determined in all fractions using the DPP3-LIA assay. Amount of DPP3 in starting material was set to 100% and remaining DPP3 amount in purification fractions was correlated to the starting material. .sup.b)Total protein amount was determined using the method of Lowry modified by Peterson (Peterson 1977. Analytical Biochemistry 356:346-356). .sup.c)Total Arg.sub.2-βNA hydrolyzing activity in μmol of substrate converted per minute was determined using the DPP3-ECA, calibrated via β-naphtylamine (0.05-100 μM). .sup.d)Purification yield was calculated form total Arg.sub.2-βNA hydrolyzing activity. Arg.sub.2-βNA hydrolyzing activity in starting material was set to 100%. .sup.e)Specific activity is defined as μmol of substrate converted per minute and mg of total protein. .sup.f)The purification factor is the quotient of specific activities after and before each purification step.

Example 4—Effect of Native DPP3 in an Animal Model

[0395] The effect of native hDPP3 injection in healthy mice was studied by monitoring the shortening fraction and renal resistive index.

[0396] Wild type Black 6 mice (8-12 weeks, group size refer to table 3) were acclimated during 2 weeks and a baseline echocardiography was done. The mice were randomly allocated to one of the two groups and, subsequently, native DPP3 protein or PBS were injected intravenously via a retro-orbital injection with a dose of 600 μg/kg for DPP3 protein.

[0397] After DPP3 or PBS injection, cardiac function was assessed by echocardiography (Gao et al. 2011) and renal function assessed by renal resistive index (Lubas et al., 2014, Dewitte et al. 2012) at 15, 60 and 120 minutes (FIG. 3).

TABLE-US-00007 TABLE 3 list of experiment groups Group Number of Animals Treatment WT + PBS 3 PBS WT + DPP3 4 Native DPP3

Results

[0398] The mice treated with native DPP3 protein show significantly reduced shortening fraction compared to the control group injected with PBS (FIG. 4A). The WT+DPP3 group also displays worsening renal function as observed by the renal resistive index increase (FIG. 4B).

Example 5—Development of Procizumab

[0399] Antibodies raised against SEQ ID NO. 2 were characterized in more detail (epitope mapping, binding affinities, specificity, inhibitory potential). Here the results for clone 1967 of SEQ ID NO.: 2 (AK1967; “Procizumab”) are shown as an example.

Determination of AK1967 Epitope on DPP3:

[0400] For epitope mapping of AK1967 a number of N- or C-terminally biotinylated peptides were synthesized (PE GmbH, Hennigsdorf, Germany). These peptides include the sequence of the full immunization peptide (SEQ ID No. 2) or fragments thereof, with stepwise removal of one amino acid from either C- or N-terminus (see table 5 for a complete list of peptides).

[0401] High binding 96 well plates were coated with 2 μg Avidin per well (Greiner Bio-One international AG, Austria) in coupling buffer (500 mM Tris-HCl, pH 7.8, 100 mM NaCl). Afterwards plates were washed and filled with specific solutions of biotinylated peptides (10 ng/well; buffer—1×PBS with 0.5% BSA). Anti-DPP3 antibody AK1967 was labelled with a chemiluminescence label according to Example 1.

[0402] The plates were filled with 200 μl of labeled and diluted detection antibody (tracer) and incubated for 4 h at room temperature. Unbound tracer was removed by washing 4 times with 350 μl washing solution (20 mM PBS, pH 7.4, 0.1% Triton X-100). Well-bound chemiluminescence was measured by using the Centro LB 960 luminometer (Berthold Technologies GmbH & Co. KG). Binding of AK1967 to the respective peptides is determined by evaluation of the relative light units (RLU). Any peptide that shows a significantly higher RLU signal than the unspecific binding of AK1967 is defined as AK1967 binder. The combinatorial analysis of binding and non-binding peptides reveals the specific DPP3 epitope of AK1967.

Determination of Binding Affinities Using Octet:

[0403] The experiment was performed using Octet Red96 (ForteBio). AK1967 was captured on kinetic grade anti-humanFc (AHC) biosensors. The loaded biosensors were then dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM). Association was observed for 120 seconds followed by 180 seconds of dissociation. The buffers used for the experiment are depicted in table 4. Kinetic analysis was performed using a 1:1 binding model and global fitting.

TABLE-US-00008 TABLE 4 Buffers used for Octet measurements Buffer Composition Assay Buffer PBS with 0.1% BSA, 0.02% Tween-21 Regeneration Buffer 10 mM Glycine buffer (pH 1.7) Neutralization Buffer PBS with 0.1% BSA, 0.02% Tween-21

Western Blot Analysis of Binding Specificity of AK1967:

[0404] Blood cells from human EDTA-blood were washed (3× in PBS), diluted in PBS and lysed by repeated freeze-thaw-cycles. The blood cell lysate had a total protein concentration of pg/ml, and a DPP3 concentration of 10 μg/ml. Dilutions of blood cell lysate (1:40, 1:80, 1:160 and 1:320) and of purified recombinant human His-DPP3 (31.25-500 ng/ml) were subjected to SDS-PAGE and Western Blot. The blots were incubated in 1.) blocking buffer (1×PBS-T with 5% skim milk powder), 2.) primary antibody solution (AK1967 1:2.000 in blocking buffer) and 3.) HRP labelled secondary antibody (goat anti mouse IgG, 1:1.000 in blocking buffer). Bound secondary antibody was detected using the Amersham ECL Western Blotting Detection Reagent and the Amersham Imager 600 UV (both from GE Healthcare).

DPP3 Inhibition Assay:

[0405] To analyze the capability of DPP3 inhibition by AK1967 a DPP3 activity assay with known procedure (Jones et al., 1982) was performed as described in example 1. The inhibitory ability AK1967 is defined as the decrease of GST-hDPP3 activity by incubation with said antibody in percent. The resulting lowered DPP3 activities are shown in an inhibition curve in FIG. 1C.

Epitope Mapping:

[0406] The analysis of peptides that AK1967 binds to and does not bind to revealed the DPP3 sequence INPETG (SEQ ID No.: 3) as necessary epitope for AK1967 binding (see table 5).

Binding Affinity:

[0407] AK1967 binds with an affinity of 2.2*10.sup.−9 M to recombinant GST-hDPP3 (kinetic curves see FIG. 5).

Specificity and Inhibitory Potential:

[0408] The only protein detected with AK1967 as primary antibody in lysate of blood cells was DPP3 at 80 kDa (FIG. 6). The total protein concentration of the lysate was 250 μg/ml whereas the estimated DPP3 concentration is about 10 μg/ml. Even though there is 25 times more unspecific protein in the lysate, AK1967 binds and detects specifically DPP3 and no other unspecific binding takes place.

[0409] AK1967 inhibits 15 ng/ml DPP3 in a specific DPP3 activity assay with an IC50 of about ng/ml (FIG. 7).

Chimerization/Humanization:

[0410] The monoclonal antibody AK1967 (“Procizumab”), with the ability of inhibiting DPP3 activity by 70%, was chosen as possible therapeutic antibody and was also used as template for chimerization and humanization.

Humanization of Murine Antibodies May be Conducted According to the Following Procedure:

[0411] For humanization of an antibody of murine origin the antibody sequence is analyzed for the structural interaction of framework regions (FR) with the complementary determining regions (CDR) and the antigen. Based on structural modelling an appropriate FR of human origin is selected and the murine CDR sequences are transplanted into the human FR. Variations in the amino acid sequence of the CDRs or FRs may be introduced to regain structural interactions, which were abolished by the species switch for the FR sequences. This recovery of structural interactions may be achieved by random approach using phage display libraries or via directed approach guided by molecular modeling (Almagro and Fransson, 2008. Humanization of antibodies. Front Biosci. 13:1619-33).

[0412] With the above context, the variable region can be connected to any subclass of constant regions (IgG, IgM, IgE. IgA), or only scaffolds, Fab fragments, Fv, Fab and F(ab)2. In example 6 and 7 below, the murine antibody variant with an IgG.sub.2a backbone was used. For chimerization and humanization a human IgG1κ backbone was used.

[0413] For epitope binding, only the CDRs are of importance. The CDRs for the heavy chain and the light chain of the murine anti-DPP3 antibody (AK1967) are shown in SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8 for the heavy chain and SEQ ID No. 9, sequence KVS and SEQ ID No. 10 for the light chain, respectively. Sequencing of the anti-DPP3 antibody (AK1967) revealed an antibody heavy chain variable region (H chain) according to SEQ ID No.: 11 and an antibody light chain variable region (L chain) according to SEQ ID No.: 12.

Example 6—Effect of Procizumab in Septic Shock-Induced Heart Failure

[0414] In this experiment, the effect of Procizumab injection in sepsis-induced heart failure rats (Rittirsch et al. 2009) was studied by monitoring the shortening fraction.

Cecal Ligation Puncture (CLP) Model of Septic Shock:

[0415] Male Wistar rats (2-3 months, 300 to 400 g, group size refers to table 6) from the Centre d′élevage Janvier (France) were allocated randomly to one of three groups. All animals were anesthetized using ketamine hydrochloride (90 mg/kg) and xylazine (9 mg/kg) intraperitoneally (i.p.). For induction of polymicrobial sepsis, CLP was performed using Rittirsch's protocol with minor modifications. A ventral midline incision (1.5 cm) was made to allow exteriorization of the cecum. The cecum is then ligated just below the ileocecal valve and punctured once with an 18-gauge needle. The abdominal cavity is then closed in two layers, followed by fluid resuscitation (3 ml/100 g body of weight of saline injected subcutaneously) and returning the animal to its cage. Sham animals were subjected to surgery, without getting their cecum punctured. CLP animals were randomized between placebo and therapeutic antibody.

Study Design:

[0416] The study flow is depicted in FIG. 8. After CLP or sham surgery the animals were allowed to rest for 20 hours with free access to water and food. Afterwards they were anesthetized, tracheotomy done and arterial and venous line laid. At 24 hours after CLP surgery either AK1967 or vehicle (saline) were administered with 5 mg/kg as a bolus injection followed by a 3 h infusion with 7.5 mg/kg. As a safety measure, hemodynamics were monitored invasively and continuously from t=0 till 3 h.

[0417] At t=0 (baseline) all CLP animals are in septic shock and developed a decrease in heart function (low blood pressure, low shortening fraction). At this time point Procizumab or vehicle (PBS) were injected (i.v.) and saline infusion was started. There were 1 control group and 2 CLP groups which are summarized in the table below (table 6). At the end of the experiment, the animals were euthanized, and organs harvested for subsequent analysis.

TABLE-US-00009 TABLE 6 list of experimental groups Group Number of Animals CLP Treatment Sham 7 No PBS CLP-PBS 6 Yes PBS CLP-PCZ 4 Yes PCZ

Invasive Blood Pressure:

[0418] Hemodynamic variables were obtained using the AcqKnowledge system (BIOPAC Systems, Inc., USA). It provides a fully automated blood pressure analysis system. The catheter is connected to the BIOPAC system through a pressure sensor.

[0419] For the procedure, rats were anesthetized (ketamine and xylazine). Animals were moved to the heating pad for the desired body temperature to 37-37.5° C. The temperature feedback probe was inserted into the rectum. The rats were placed on the operating table in a supine position. The trachea was opened and a catheter (16G) was inserted for an external ventilator without to damage carotid arteries and vagus nerves. The arterial catheter was inserted into the right carotid artery. The carotid artery is separate from vagus before ligation. A central venous catheter was inserted through the left jugular vein allowing administration of PCZ or PBS.

[0420] Following surgery, the animals were allowed to rest for the stable condition prior to hemodynamic measurements. Then baseline blood pressure (BP) were recorded. During the data collection, saline infusion via arterial line was stopped.

Echocardiography:

[0421] Animals were anesthetized using ketamine hydrochloride. Chests were shaved and rats were placed in decubitus position. For transthoracic echocardiographic (TTE) examination a commercial GE Healthcare Vivid 7 Ultra-sound System equipped with a high frequency (14-MHz) linear probe and 10-MHz cardiac probe was used. All examinations were recorded digitally and stored for subsequent off-line analysis.

[0422] Grey scale images were recorded at a depth of 2 cm. Two-dimensional examinations were initiated in a parasternal long axis view to measure the aortic annulus diameter and the pulmonary artery diameter. M-mode was also employed to measure left ventricular (LV) dimensions and assess fractional shortening (FS %). LVFS was calculated as LV end-diastolic diameter—LV end-systolic diameter/LV end-diastolic diameter and expressed in %. The time of end-diastole was therefore defined at the maximal diameter of the LV. Accordingly, end-systole was defined as the minimal diameter in the same heart cycle. All parameters were measured manually. Three heart cycles were averaged for each measurement.

[0423] From the same parasternal long axis view, pulmonary artery flow was recorded using pulsed wave Doppler. Velocity time integral of pulmonary artery outflow was measured.

[0424] From an apical five-chamber view, mitral flow was recorded using pulsed Doppler at the level of the tip of the mitral valves.

Results:

[0425] The septic shock-induced heart failure rats treated with PBS (CLP+PBS) show reduced shortening fraction compared to the sham animals (FIG. 9A). The CLP+PBS group also displays high mortality rate (FIG. 9B). In contrast, application of Procizumab to sep-induced heart failure rats improves shortening fraction (FIG. 9A) and drastically reduces the mortality rate (FIG. 9B).

Example 7—Effect of Procizumab on Heart and Kidney Function

[0426] The effect of Procizumab in isoproterenol-induced heart failure in mice was studied by monitoring the shortening fraction and renal resistive index.

Isoproterenol-Induced Cardiac Stress in Mice:

[0427] Acute heart failure was induced in male mice at 3 months of age by two daily subcutaneous injections of 300 mg/kg of Isoproterenol, a non-selective β-adrenergic agonist (DL-Isoproterenol hydrochloride, Sigma Chemical Co) (ISO) for two days (VerParo et al. 2016). ISO dilution was performed in NaCl 0.9%. ISO-treated mice were randomly assigned to two groups (Table 7) and PBS or Procizumab (10 mg/kg) were injected intravenously after baseline echocardiography (Gao et al., 2011) and renal resistive index measurements (Lubas et al., 2014, Dewitte et al, 2012) were performed at day 3 (FIGS. 10 A and B). Cardiac function was assessed by echocardiography (Gao et al., 2011) and by the renal resistive index (Lubas et al., 2014, Dewitte et al., 2012) at 1 hour, 6 hours and 24 hours (FIGS. 10 A and B). The group of mice that was injected with vehicle (PBS) instead of isoproterenol was subjected to no further pharmacological treatment and served as the control group (Table 7).

TABLE-US-00010 TABLE 7 list of experimental groups Group Number of Animals Treatment Sham + PBS 27 PBS HF + PBS 15 PBS HF + PCZ 20 Procizumab (PCZ)

Results:

[0428] Application of Procizumab to isoproterenol-induced heart failure mice restores heart function within the first hour after administration (FIG. 11A). Kidney function of sick mice shows significant improvement at 6 hours post Procizumab injection and is comparable to the kidney function of sham animals at 24 hours (FIG. 11B).

Example 8—DPP3 Indicates Vasopressor Need and Response to Vasopressor Therapy

[0429] DPP3 concentrations in plasma of septic shock patients were determined using a hDPP3 immunoassay and related to the need for vasopressor therapy.

Study Cohort—Septic Shock

[0430] Plasma samples from 292 patients that were diagnosed with septic shock from the AdrenOSS (see example 2) study were screened for DPP3. Human DPP3 was measured as described in Example 1.

Results

[0431] Patients with an increased need to vasopressor administration for more than 5 days had high plasma concentrations of DPP3 (FIG. 12A). In contrast, patients that responded to vasopressor administration and where vasopressor administration could be discontinued within the first 5 days, had significantly reduced plasma DPP3 concentrations (FIG. 12B). This indicates that development of refractory shock and thus an increased need of vasopressor administration due to a diminished response of this therapy, is related to high DPP3 plasma concentrations in patients with septic shock.

[0432] Patients with vasopressor-resistant refractory septic shock (noradrenaline >0.5 μg/kg/min) show significantly higher plasma DPP3 concentrations than compared to patients that required noradrenaline doses of <0.5 μg/kg/min (p<0.001) (FIG. 16A). Moreover, the DPP3 plasma concentration was strongly associated with mortality in patients with vasopressor-resistant refractory septic shock (FIG. 16B).

Example 9—DPP3 is Related to Refractory Shock

[0433] DPP3 concentrations in plasma of cardiogenic shock patients were determined using a hDPP3 immunoassay and related to the development of refractory shock.

Study Cohort—Cardiogenic Shock

[0434] Plasma samples from 57 patients that were diagnosed with cardiogenic shock after acute myocardial infarction were screened for DPP3. Human DPP3 was measured as described in Example 1.

Results

[0435] Patients with a DPP3 plasma concentration above a certain threshold on admission (59.1 ng/ml; 3.sup.rd quartile) developed refractory shock to a higher extent (47%) than patients with DPP3 plasma concentrations below 59.1 ng/ml (12%) (FIG. 13).

Example 10—DPP3 and Bio-ADM Indicate Short-Term Mortality in Shock

[0436] DPP3 and bio-ADM concentrations in plasma of septic shock patients were determined using a hDPP3 and a bio-ADM immunoassay and related to the short term-mortality of the patients.

Study Cohort—Septic Shock

[0437] Plasma samples from 292 patients that were diagnosed with septic shock from the AdrenOSS study were screened for DPP3 and bio-ADM. Human DPP3 was measured as described in Example 1. Bio-ADM was measured as described in Weber et al. (Weber et al. 2017. JALM 2(2): 1-4).

Results

[0438] Plasma concentrations of bio-ADM and DPP3 were measured in septic shock patients. Patients were grouped according to specific cut-offs determined to be the 3.sup.rd quartile of all measured plasma concentrations of the respective marker (Table 8). Equal numbers of patients had either high DPP3 only or high bio-ADM only (15.4%), while a lesser extent showed elevated plasma concentrations in both, bio-ADM and DPP3 (9.6%).

TABLE-US-00011 TABLE 8 Patient numbers with low/high DPP3 and low/high bio-ADM. Cut- offs were assignee based on the Q3 (highest 25%) for both. DPP3 low DPP3 high (<48.4 ng/ml) (>48.4 ng/ml) bio-ADM low (<213 pg/mL) 174 (59.6%) 45 (15.4%) bio-ADM high (>213 pg/mL)  45 (15.4%) 28 (9.6%) 

[0439] Mortality within the first 4 weeks after admission was related to the bio-ADM and DPP3 concentration on admission. Patients with either only a high bio-ADM or only a high DPP3 plasma concentration had a substantial increased risk to die within the first 4 weeks compared to patients that had a plasma concentration of either bio-ADM (FIG. 14A) or either DPP3 (FIG. 14B) below a certain threshold (3.sup.rd quartile). Survival in patients with high bio-ADM was better than survival in patients with high DPP3.

[0440] When both markers, bio-ADM and DPP3, were combined, an even higher risk for short-term mortality was identified in comparison to the mortality that was related to an increase of one of both markers only (FIG. 14C). Merely 28.6% of the patients with high bio-ADM and high-DPP3 plasma concentrations survived the first 4 weeks after admission.

Example 11—DPP3 and Bio-ADM DPP3 Indicates Vasopressor Need and Response to Vasopressor Therapy

[0441] DPP3 and bio-ADM concentrations in plasma of septic shock patients were determined using a hDPP3 and a bio-ADM immunoassay and related to the need for vasopressor therapy.

Study Cohort—Septic Shock

[0442] In 292 plasma samples from patients that were diagnosed with septic shock from the AdrenOSS study DPP3 and bio-ADM concentrations were measured. Human DPP3 was measured as described in Example 1. Bio-ADM was measured as described in Weber et al. (Weber et al. 2017. JALM 2(2): 1-4).

Results

[0443] Plasma concentrations of bio-ADM and DPP3 were measured in septic shock patients. Patients were grouped according to specific cut-offs determined as the 3.sup.rd quartile of all measured plasma concentrations of the respective marker in the septic shock cohort (low DPP3≤48.4 ng/mL, low bio-ADM<213 pg/mL; high bio-ADM≥213 pg/mL; high DPP3≥48.4 ng/mL). Patients with high DPP3, but low bio-ADM plasma concentrations had a higher need for consecutive vasopressor administration compared to patients with either low DPP3+low bio-ADM or low DPP3+high bio-ADM (FIG. 15; Table 9). In contrast, patients that had low DPP3 and low bio-ADM plasma concentrations, or patients that had low DPP3, but high bio-ADM plasma concentrations on admission could be discontinued of vasopressor administration earlier than patients with high DPP3 plasma concentrations on admission (FIG. 15; Table 9). This indicates that vasopressor therapy in septic shock patients with high bio-ADM can be discontinued earlier due to a better therapeutic response compared to septic shock patients with high DPP3. These patients (high DPP3) need longer treatment with vasopressors due to non-responsiveness.

TABLE-US-00012 TABLE 9 Patients with vasopressor need grouped according to their DPP3 and bio-ADM plasma concentration at admission. Cut- offs were assigned based on the Q3 (highest 25% of determined values) for both; low DPP3 < 48.4 ng/mL, low bio-ADM < 213 pg/mL; high bio-ADM ≥ 213 pg/mL; high DPP3 ≥ 48.4 ng/mL; 7: ≥7 days on vasopressor or dead within 7 days. Days with vasopres- DPP3 Low DPP3 low DPP3 high DPP3 high sor need bioADM low bioADM high bioADMlow bioADM high 1 42 8 5 2 2 49 11 6 2 3 25 6 8 2 4 16 4 1 2 5 8 3 2 0 6 3 1 6 1 7 31 12 17 19

EXAMPLES OF SEQUENCES

[0444]

TABLE-US-00013 hDPP3 aa 1-737 SEQ ID No. 1 MADTQYILPNDIGVSSLDCREAFRLLSPTERLYAYHLSRAAWYGGLAVLLQTSPEAP YIYALLSRLFRAQDPDQLRQHALAEGLTEEEYQAFLVYAAGVYSNMGNYKSFGDTK FVPNLPKEKLERVILGSEAAQQHPEEVRGLWQTCGELMFSLEPRLRHLGLGKEGITTY FSGNCTMEDAKLAQDFLDSQNLSAYNTRLFKEVDGEGKPYYEVRLASVLGSEPSLDS EVTSKLKSYEFRGSPFQVTRGDYAPILQKVVEQLEKAKAYAANSHQGQMLAQYIESF TQGSIEAHKRGSRFWIQDKGPIVESYIGFIESYRDPFGSRGEFEGFVAVVNKAMSAKFE RLVASAEQLLKELPWPPTFEKDKFLTPDFTSLDVLTFAGSGIPAGINIPNYDDLRQTEG FKNVSLGNVLAVAYATQREKLTFLEEDDKDLYILWKGPSFDVQVGLHELLGHGSGK LFVQDEKGAFNFDQETVINPETGEQIQSWYRSGETWDSKFSTIASSYEECRAESVGLY LCLHPQVLEIFGFEGADAEDVIYVNWLNMVRAGLLALEFYTPEAFNWRQAHMQARF VILRVLLEAGEGLVTITPTTGSDGRPDARVRLDRSKIRSVGKPALERFLRRLQVLKSTG DVAGGRALYEGYATVTDAPPECFLTLRDTVLLRKESRKLIVQPNTRLEGSDVQLLEY EASAAGLIRSFSERFPEDGPELEEILTQLATADARFWKGPSEAPSGQA hDPP3 aa 474-493 (N-Cys)-immunization peptide with  additional N-terminal Cystein SEQ ID No. 2 CETVINPETGEQIQSWYRSGE hDPP3 aa 477-482-epitope of AK1967 SEQ ID No. 3 INPETG variable region of murine AK1967 in heavy chain  SEQ ID No. 4 QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMSVGWIRQPSGKGLEWLAHIWWNDN KSYNPALKSRLTISRDTSNNQVFLKIASVVTADTGTYFCARNYSYDYWGQGTTLTVSS variable region of murine AK1967 in light chain  SEQ ID No. 5 DVVVTQTPLSLSVSLGDPASISCRSSRSLVHSIGSTYLHWYLQKPGQSPKLLIYKVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK CDR1 of murine AK1967 in heavy chain  SEQ ID No. 6 GFSLSTSGMS CDR2 of murine AK1967 in heavy chain  SEQ ID No. 7 IWWNDNK CDR3 of murine AK1967 in heavy chain  SEQ ID No. 8 ARNYSYDY CDR1 of murine AK1967 in light chain  SEQ ID No. 9 RSLVHSIGSTY CDR2 of murine AK1967 in light chain  KVS CDR3 of murine AK1967 in light chain  SEQ ID No. 10 SQSTHVPWT humanized AK1967-heavy chain sequence (IgG1κ backbone) SEQ ID No. 11 MDPKGSLSWRILLFLSLAFELSYGQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGMS VGWIRQPPGKALEWLAHIWWNDNKSYNPALKSRLTITRDTSKNQVVLTMTNMDPV DTGTYYCARNYSYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG humanized AK1967-light chain sequence (IgG1κ backbone) SEQ ID No. 12 METDTLLLWVLLLWVPGSTGDIVMTQTPLSLSVTPGQPASISCKSSRSLVHSIGSTYLY WYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQST HVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC Angiotensin II (synonyme: 5-isoleucine-angiotensin II) SEQ ID No. 13 DRVYIHPF Angiotensin II analogue (5-valine-angiotensin II) SEQ ID No. 14 DRVYVHPF Angiotensin II analogue (Asn.sup.1-Phe.sup.4) SEQ ID No. 15 NRVFIHPF Angiotensin II hexapeptide SEQ ID No. 16 VYIHPF Angiotensin II nonapeptide SEQ ID No. 17 NRVYYVHPF Angiotensin II analogue ([Asn.sup.1-Ile.sup.5-Ile.sup.8]-Angiotensin II) SEQ ID No. 18 NRVYIHPI Angiotensin II analogue ([Asn.sup.1-Ile.sup.5-Ala.sup.8]-angiotensin II) SEQ ID No. 19 NRVYIHPA Angiotensin II analogue ([Asn.sup.1-diiodoTyr.sup.4-Ile.sup.5]-angiotensin II  SEQ ID No. 20 NRVYIHPF Angiotensin III SEQ ID No. 21 RVYIHPF Angiotensin III analogue (Val.sup.4-Angiotensin III) SEQ ID No. 22 RVYVHPF Angiotensin III analogue (Phe.sup.3-angiotensin III) SEQ ID No. 23 RVFIHPF Angiotensin III analogue ([Ile.sup.4-Ala.sup.7]-angiotensin III) SEQ ID No. 24 RVYIHPA Angiotensin III analogue (diiodoTyr.sup.3-Ile.sup.4]-angiotensin III) SEQ ID No. 25 RVYIHPF Angiotensin IV SEQ ID No. 26 VYIHPF Angiotensin IV analogue (Val.sup.3-angiotensin IV) SEQ ID No. 27 VYVHPF Angiotensin IV analogue (Phe.sup.2-angiotensin IV) SEQ ID No. 28 VFIHPF Angiotensin IV analogue ([Ile.sup.3-Ala.sup.6]-angiotensin IV) SEQ ID No. 29 VYIHPA Angiotensin IV analogue ([diiodoTyr.sup.2-Ile.sup.3-angiotensin IV) SEQ ID No. 30 VYIHPF (proADM): 164 amino acids (22-185 of preproADM) SEQ ID No. 31 ARLDVASEF RKKWNKWALS RGKRELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL (Proadrenomedullin N-20 terminal peptide, PAMP):  amino acids 22-41 of preproADM SEQ ID No. 32 ARLDVASEFRKKWNKWALS R (Midregional proAdrenomedullin, MR-proADM):  amino acids 45-92 of preproADM SEQ ID No. 33 ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV (mature Adrenomedullin (matureA DM); amidated ADM; bio-ADM; hADM): amino acids 95-146-CONH.sub.2 SEQ ID No. 34 YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGY-CONH.sub.2 (Adrenomedullin 1-52-Gly (ADM 1-52-Gly)):  amino acids 95-147 of preproADM SEQ ID No. 35 YRQSMN NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYG (C-terminal proAdrenomedullin, CT-proADM):  amino acids 148-185 of preproADM SEQ ID No. 36 RRR RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL (N-terminal part of mature ADM): amino acids 1-21 of mature ADM SEQ ID No. 37 YRQSMNNFQGLRSFGCRFGTC (CDR1 heavy chain anti-ADM antibody) SEQ ID No. 38 GYTFSRYW (CDR2 heavy chain anti-ADM antibody) SEQ ID No. 39 ILPGSGST (CDR3 heavy chain anti-ADM antibody) SEQ ID No. 40 TEGYEYDGFDY (CDR1 light chain anti-ADM antibody) SEQ ID No. 41 QSIVYSNGNTY (CDR3 light chain anti-ADM antibody) SEQ ID No. 42 FQGSHIPYT (anti-ADM antibody (Adrecizumab) heavy chain) SEQ ID No. 43 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSRYWIEWVRQAPGQGLEWIGEILPGSGS TNYNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCTEGYEYDGFDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (anti-ADM antibody (Adrecizumab) light chain) SEQ ID NO: 44 DVVLTQSPLSLPVTLGQPASISCRSS LEWYLQRPGQSPRLLIY NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC  FGGGTKLEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

FIGURE DESCRIPTION

[0445] FIG. 1: Kaplan-Meier survival plots in relation to low (<40.5 ng/mL) and high (≥40.5 ng/mL) DPP3 concentrations. (A) 7-Day survival of patients with sepsis in relation to DPP3 plasma concentration; (B) 7-Day survival of Patients with cardiogenic shock in relation to DPP3 plasma concentration; (C) 7-Day survival of patients with septic shock in relation to DPP3 plasma concentration.

[0446] FIG. 2: SDS-PAGE on a gradient gel (4-20%) of native hDPP3 purified from human erythrocyte lysate. Molecular weight marker is indicated as arrows.

[0447] FIG. 3: Experimental design—Effect of native DPP3 in an animal model.

[0448] FIG. 4: (A) DPP3 injection causes shortening fraction reduction and therefore leads to deteriorating heart function. (B) Decreased kidney function is also observed via increased renal resistive index.

[0449] FIG. 5: Association- and dissociation curve of the AK1967-DPP3 binding analysis using Octet. AK1967 loaded biosensors were dipped into a dilution series of recombinant GST-tagged human DPP3 (100, 33.3, 11.1, 3.7 nM) and association and dissociation monitored.

[0450] FIG. 6: Western Blot of dilutions of blood cell lysate and detection of DPP3 with AK1967 as primary antibody.

[0451] FIG. 7: Inhibition curve of native DPP3 from blood cells with inhibitory antibody AK1967. Inhibition of DPP3 by a specific antibody is concentration dependent, with an IC.sub.50 at ˜15 ng/ml when analyzed against 15 ng/ml DPP3.

[0452] FIG. 8: Experimental setup—Effect of Procizumab in sepsis-induced heart failure.

[0453] FIG. 9: Procizumab drastically improves shortening fraction (A) and mortality rate (B) in sepsis-induced heart failure rats.

[0454] FIG. 10: Experimental design—Isoproterenol-induced cardiac stress in mice followed by Procizumab treatment (B) and control (A).

[0455] FIG. 11: Procizumab improved shortening fraction (A) and reduced the renal resistive index (B) within 1 hour and 6 hours after administration, respectively, in isoproterenol-induced heart failure mice.

[0456] FIG. 12: (A) Days with need of vasopressor therapy in septic shock patients in relation to DPP3 plasma concentrations. ≥7 days on vasopressor or dead within 7 days; * p<0.05 in post hoc comparison vs. groups 1-4 (B) Need of vasopressor therapy in septic shock patients in relation to DPP3 plasma concentrations. Patients with vasopressor therapy for maximal 5 days (≤5), and patients with vasopressor therapy longer than 5 days or that died within 7 days were grouped together (>5).

[0457] FIG. 13: DPP3 is related to refractory shock. High DPP3 concentrations in plasma of cardiogenic shock patients are related with a higher risk to develop refractory cardiogenic shock compared to patients that have DPP3 plasma concentrations below a certain threshold.

[0458] FIG. 14: Kaplan-Meier survival plots (A) 4-week survival of patients with septic shock in relation to bio-ADM plasma concentration; values are grouped in quartiles (B) 4-week survival of patients with septic shock in relation to DPP3 plasma concentration; values are grouped in quartiles (C) 4-week survival of patients with septic shock in relation to bio-ADM and DPP3 plasma concentration; Cut-offs were determined based on 3.sup.rd quartile of all measured plasma concentrations of the respective marker: both low DPP3<48.4 ng/mL, bio-ADM<213 pg/mL; bio-ADM high≥213 pg/mL; DPP3 high≥48.4 ng/mL.

[0459] FIG. 15: Patients with vasopressor need grouped according to their DPP3 and bio-ADM plasma concentration on admission. Proportions of patients receiving vasopressors for 1-7 days are shown in greyscale—lighter colors represent longer treatment duration. Cut-offs were assigned based on the Q3 (highest 25% of determined values) for both; low DPP3<48.4 ng/mL, low bio-ADM<213 pg/mL; high bio-ADM≥213 pg/mL; high DPP3≥48.4 ng/mL; 7:≥7 days on vasopressor or dead within 7 days.

[0460] FIG. 16: (A) Plasma DPP3 concentration in patients with septic refractory shock who are in need of the vasopressor noradrenalin below (n=186) and above (n=95) 0.5 μg/kg/min (p<0.001). (B) Kaplan-Meier survival plots (A) 4-week survival of patients with septic refractory shock in relation to DPP3 plasma concentration; values are grouped in quartiles.

REFERENCES

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